Viruses usurp host cell functions and recondition the cell to optimize it for viral replication. They do this by redirecting host proteins and by bringing in their own proteins as necessary. To understand the underlying mechanisms, it is important to characterize viral events in the context of the cell and identify viral and host factors involved in these process. We have been able to apply whole cell electron cryotomography (ECT) to follow the assembly and maturation of Sulfolobus turreted icosahedral virus (STIV) in a life-like state in intact archaeon Sulfolobus cells. The whole cell ECT approach, which is limited in most systems due to the cell thickness, makes STIV/ Sulfolobus one of the few model systems to correlate the structural changes that occur in an entire cell as a consequence of virus infection in 3D at low-nm resolution. We propose to combine ECT, single particle CryoEM reconstruction, genetic and biochemical approaches to (1) determine the morphogenesis of STIV, to (2) dissect the factors contributing to the distinct cellular distribution of the particles at different maturation stages, and to (3) characterize a new viral release mechanism mediated by infection-induced pyramid structures. Cellular tomography studies have revealed DNA-filled STIV virions, DNA-free procapsids and partially assembled particles and showed that the capsid and inner membrane co-assemble in the cytoplasm of Sulfolobus that lacks membrane-containing organelles. The assembled procapsids are packaged with DNA and mature to virions. In Specific Aim 1, we propose to identify the specialized packaging vertex and characterize the structural changes accompanying packaging. We also found that STIV virions tend to form quasi-crystalline arrays in the cell while procapsids are mostly scattered outside or on the edges of the arrays. We hypothesize that the particles are organized to form viral arrays when packaging the genome. We propose to test the hypothesis by uncoupling capsid assembly and DNA packaging. The model predicts that blocking DNA-packaging will block array formation. Furthermore, we found that viral infection induced pyramid-like protrusions, which have sharply defined facets and apexes. Forming these pyramids requires substantial remodeling of the cellular membrane and destruction of the surface protein layer, leading to insights about this new virus-release mechanism. We will characterize the role of viral protein C92 (a major pyramid protein) in pyramid formation and pyramid-mediated viral release. The proposal will contribute significantly to our understanding of the cell biology of archaeal virus pathogenesis and viruses in the PRD1-Adeno lineage in general. PUBLIC HEALTH RELEVANCE: Viruses cause many human diseases and agricultural loses. To understand how viruses replicate and hijack hosts, it is important to follow viral events in vivo and characterize viral-host interactions involved in various steps of viral life cycle. The proposal will contribute significantly to our understanding of the cell biology of Sulfolobus turreted icosahedral virus infection and viruses in the PRD1-Adeno lineage in general.